O-GlcNAcylation Levels Research Articles

An overview of tools to decipher O-GlcNAcylation from historical approaches to new insights.

O-GlcNAcylation is a post-translational modification which affects approximately 5000 human proteins. Its involvement has been shown in many if not all biological processes. Variations in O-GlcNAcylation levels can be associated with the development of diseases. Deciphering the role of O-GlcNAcylation is an important issue to (i) understand its involvement in pathophysiological development and (ii) develop new therapeutic strategies to modulate O-GlcNAc levels. Over the past 30 years, despite the development of several approaches, knowledge of its role and regulation have remained limited. This review proposes an overview of the currently available tools to study O-GlcNAcylation and identify O-GlcNAcylated proteins. Briefly, we discuss pharmacological modulators, methods to study O-GlcNAcylation levels and approaches for O-GlcNAcylomic profiling. This review aims to contribute to a better understanding of the methods used to study O-GlcNAcylation and to promote efforts in the development of new strategies to explore this promising modification.

RNA binding motif protein 3 (RBM3) promotes protein kinase B (AKT) activation to enhance glucose metabolism and reduce apoptosis in skeletal muscle of mice under acute cold exposure.

The main danger of cold stress to animals in cold regions is systemic metabolic changes and protein synthesis inhibition. RBM3, an exceptional cold shock protein, is rapidly upregulated in response to hypothermia to resist the adverse effects of cold stress. However, the mechanism of the protective effect and the rapid upregulation of RBM3 remains unclear. O-GlcNAcylation, an atypical O-glycosylation, is precisely regulated only by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) and participates in the signal transduction of multiple cellular stress responses as a "stress and nutrition receptor." Therefore, our study aimed to explore the mechanism of RBM3 regulating glucose metabolism and promoting survival in skeletal muscle under acute cold exposure. Meanwhile, our study verifies whether O-GlcNAcylation mediated by OGT rapidly upregulates RBM3. The blood and skeletal muscle of mice were collected at the end of cold exposure treatment for 0, 2, and 4h. Changes in levels of RBM3, AKT, glycolysis apoptosis, and OGT were measured. The results show that acute cold exposure upregulated RBM3, OGT, and AKT phosphorylation and increased energy consumption, which enhanced glycolysis and prevent apoptosis. In the 32°C mild hypothermia model in vitro, overexpression of RBM3 enhanced AKT phosphorylation. Meanwhile, inactivation of AKT by wortmannin resulted in increased apoptosis and decreased glucose metabolism in skeletal muscle under acute cold exposure. In addition, OGT-mediated O-GlcNAcylation of p65 was confirmed in mouse myoblast cell line (C2C12) cells at mild hypothermia. O-GlcNAcylation level affected p65 activity and nuclear translocation. Compared with wild type (WT) mice, RBM3 and p65 phosphorylation were decreased in specific skeletal muscle Ogt (KO) mice, whereas AKT phosphorylation, glycolysis, and apoptosis were increased. Taken together, O-GlcNAcylation of p65 upregulates RBM3 to promote AKT phosphorylation, enhance glucose metabolism, and reduce apoptosis in skeletal muscle of mice under acute cold exposure.

Heat-Shock Triggers Inverted Induction of Hypo-S-Nitrosylation and Hyper-O-GlcNAcylation.

Protein S-nitrosylation (SNO) and O-GlcNAcylation are important posttranslational modifications. The biological connection between SNO and O-GlcNAcylation is not clear. We aim to identify the crosstalk between SNO and O-GlcNAcylation during heat-shock. Ex vivo heat-shock on mouse tissues together with in vitro heat-shock on culture cells was performed and global levels of SNO and O-GlcNAcylation were analyzed with Biotin-switch assay (BSA) and RL2 immunoblots. Heat-shock induces hypo-SNO in parallel with hyper-O-GlcNAcylation. Inverted induction of hypo-SNO and hyper-O-GlcNAcylation is globally progressed in a time-dependent manner. Moreover, heat-shock ubiquitously facilitates S-denitrosylation (SdeNO) of endogenous SNO-proteins including SNO-OGT, SNO-Hsp70, SNO-Hsp90, SNO-Akt, and SNOactin. Particularly, SdeNO of SNO-OGT leads to enhanced OGT activity. These findings provide mechanistic evidence that heat-shock triggers SdeNO of SNOOGT by which OGT activity is up-regulated, resulting in hyper-O-GlcNAcylation.

Tripeptide Leu-Pro-Phe from Corn Protein Hydrolysates Attenuates Hyperglycemia-Induced Neural Tube Defect in Chicken Embryos.

Neural tube defect (NTD) is the most common and severe embryopathy causing embryonic malformation and even death associated with gestational diabetes mellitus (GDM). Leu-Pro-Phe (LPF) is an antioxidative tripeptide isolated from hydrolysates of corn protein. However, the biological activity of LPF in vivo and in vitro remains unclear. This study is aimed at investigating the protective effects of tripeptide LPF against NTD in the high glucose exposure condition and delineate the underlying biological mechanism. We found that LPF alleviated NTD in the high glucose-exposed chicken embryo model. In addition, DF-1 chicken embryo fibroblast was loaded with high glucose for induction of oxidative stress and abnormal O-GlcNAcylation in vitro. LPF significantly decreased accumulation of reactive oxygen species and content of malondialdehyde in DF-1 cells but increased the ratio of reduced glutathione and oxidized glutathione in chick embryo. Oxygen radical absorbance capacity results showed that LPF itself had good free radical scavenging capacity and could enhance antioxidant activity of the cell content. Mechanistic studies suggested that the resistance of LPF to oxidative damage may be related to promotion of NRF2 expression and nuclear translocation. LPF alleviated the overall O-GlcNAcylation level of cellular proteins under high glucose conditions and restored the level of Pax3 protein. Collectively, our findings indicate that LPF peptide could act as a nutritional supplement for the protection of development of embryonic neural tube affected by GDM.

Abstract P3014: Dislocation Of Hexokinase1 From Mitochondria Results In Heart Failure With Preserved Ejection Fraction Through Hyper O-glcnacylation In Endothelial Cells.

Introduction: It is reported that endothelial cell (EC) dysfunction underlies the pathogenesis of heart failure with preserved ejection fraction (HFpEF). EC are known to highly rely on glycolysis to keep their function, but the metabolism of EC under the situation with HFpEF is poorly understood. Objective: We sought to elucidate the role of hexokinase 1 (HK1) in ECs for the development of HFpEF. Results: Isolated ECs from mouse hearts showed higher protein expression of HK1 than isolated cardiomyocytes and fibroblasts, suggesting HK1 has an important role in EC function. Immunogold-staining of HK1 in hearts from C57BL6 mice treated with high fat diet and LNAME, a mouse model of HFpEF, showed increased dislocation of HK1 from the mitochondria in ECs. To study the role of HK1 dislocation, we generated ΔE1HK1 mice with mitochondrial-binding domain of the endogenous HK1 replaced with Flag tag. Subcellular fractionation confirmed that HK1 was dislocated from mitochondria in these mice. Echocardiography showed that the mice developed impaired cardiac relaxation at 20 weeks of age and HFpEF at 40 weeks of age. Significant increased fibrosis and microvascular rarefaction (MR) were observed at 40 weeks of age, but only MR was observed at 20 weeks of age, suggesting EC dysfunction likely precedes and promotes the development of HFpEF in these mice. To study the angiogenic ability of ECs with HK1 dislocation, we isolated ECs from ΔE1HK1 hearts and performed a tubing assay, and observed significantly less tubing in ΔE1HK1 ECs. To elucidate the mechanism by which angiogenesis is reduced in ΔE1HK1 ECs, we next performed metabolomics analysis in these cells. Our data indicated that the levels of metabolites in hexosamine-biosynthetic pathway (HBP) were altered between wild-type (WT) and ΔE1HK1 EC. We next analyzed the levels of O-GlcNAcylation, and showed that ECs from ΔE1HK1 hearts have higher O-GlcNAcylation than those from WT. Finally, treatment with ST045849, an inhibitor of O-GlcNAc transferase (OGT), rescued the less angiogenic ability in ECs from ΔE1HK1 hearts. Conclusion: Our studies demonstrate that dislocation of HK1 plays an important role in the development of HFpEF through hyper-O-GlcNAcylation. Drugs that inhibit OGT may provide a therapeutic option for HFpEF.

O-GlcNAcylation promotes pancreatic tumor growth by regulating malate dehydrogenase 1.

Oncogenic Kras-activated pancreatic ductal adenocarcinoma (PDAC) cells highly rely on an unconventional glutamine catabolic pathway to sustain cell growth. However, little is known about how this pathway is regulated. Here we demonstrate that Kras mutation induces cellular O-linked β-N-acetylglucosamine (O-GlcNAc), a prevalent form of protein glycosylation. Malate dehydrogenase 1 (MDH1), a key enzyme in the glutamine catabolic pathway, is positively regulated by O-GlcNAcylation on serine 189 (S189). Molecular dynamics simulations suggest that S189 glycosylation on monomeric MDH1 enhances the stability of the substrate-binding pocket and strengthens the substrate interactions by serving as a molecular glue. Depletion of O-GlcNAcylation reduces MDH1 activity, impairs glutamine metabolism, sensitizes PDAC cells to oxidative stress, decreases cell proliferation and inhibits tumor growth in nude mice. Furthermore, O-GlcNAcylation levels of MDH1 are elevated in clinical PDAC samples. Our study reveals that O-GlcNAcylation contributes to pancreatic cancer growth by regulating the metabolic activity of MDH1.

Protein O-GlcNAcylation in cardiovascular diseases.

O-GlcNAcylation is a post-translational modification of protein in response to genetic variations or environmental factors, which is controlled by two highly conserved enzymes, i.e. O-GlcNAc transferase (OGT) and protein O-GlcNAcase (OGA). Protein O-GlcNAcylation mainly occurs in the cytoplasm, nucleus, and mitochondrion, and it is ubiquitously implicated in the development of cardiovascular disease (CVD). Alterations of O-GlcNAcylation could cause massive metabolic imbalance and affect cardiovascular function, but the role of O-GlcNAcylation in CVD remains controversial. That is, acutely increased O-GlcNAcylation is an adaptive heart response, which temporarily protects cardiac function. While it is harmful to cardiomyocytes if O-GlcNAcylation levels remain high in chronic conditions or in the long run. The underlying mechanisms include regulation of transcription, energy metabolism, and other signal transduction reactions induced by O-GlcNAcylation. In this review, we will focus on the interactions between protein O-GlcNAcylation and CVD, and discuss the potential molecular mechanisms that may be able to pave a new avenue for the treatment of cardiovascular events.

PGM3 regulates beta-catenin activity to promote colorectal cancer cell progression.

The hexosamine biosynthetic pathway (HBP) is connected to abnormal N- and O-linked protein glycosylation in cancer, which performs critical roles in tumorigenesis. However, the regulation mechanisms of HBP and its role in colorectal cancer (CRC) progression remain unexplained. This study analyzed the expression level of phosphoglucomutase 3 (PGM3), a key enzyme in HBP, and identified its function in CRC cell lines. Analysis of publicly available CRC microarray data determined that PGM3 is upregulated in CRC tumor tissues. Furthermore, functional experiments emphasized the significant roles of PGM3 in facilitating CRC cell proliferation and migration. Mechanistically, we demonstrated that the activity of β-catenin in CRC was maintained by PGM3-mediated O-GlcNAcylation. PGM3 knockdown or inhibition of O-GlcNAc transferase decreased β-catenin activity and the expression levels of its downstream targets. Collectively, our findings indicate that PGM3 exhibits tumor-promoting roles by elevating O-GlcNAcylation level and maintaining β-catenin activity, and might serve as a prognostic biomarker and treatment target in CRC.

O-GlcNAcylation: An Emerging Protein Modification Regulating the Hippo Pathway.

Simple SummaryThe contact point between the Hippo pathway, which serves as a central hub for various external environments, and O-GlcNAcylation, which is a non-canonical glycosylation process acting as a dynamic regulator in various signal transduction pathways, has recently been identified. This review aims to summarize the function of O-GlcNAcylation as an intrinsic and extrinsic regulator of the Hippo pathway.The balance between cellular proliferation and apoptosis and the regulation of cell differentiation must be established to maintain tissue homeostasis. These cellular responses involve the kinase cascade-mediated Hippo pathway as a crucial regulator. Hence, Hippo pathway dysregulation is implicated in diverse diseases, including cancer. O-GlcNAcylation is a non-canonical glycosylation that affects multiple signaling pathways through its interplay with phosphorylation in the nucleus and cytoplasm. An abnormal increase in the O-GlcNAcylation levels in various cancer cells is a potent factor in Hippo pathway dysregulation. Intriguingly, Hippo pathway dysregulation also disrupts O-GlcNAc homeostasis, leading to a persistent elevation of O-GlcNAcylation levels, which is potentially pathogenic in several diseases. Therefore, O-GlcNAcylation is gaining attention as a protein modification that regulates the Hippo pathway. This review presents a framework on how O-GlcNAcylation regulates the Hippo pathway and forms a self-perpetuating cycle with it. The pathological significance of this self-perpetuating cycle and clinical strategies for targeting O-GlcNAcylation that causes Hippo pathway dysregulation are also discussed.

Global O-GlcNAcylation changes impact desmin phosphorylation and its partition toward cytoskeleton in C2C12 skeletal muscle cells differentiated into myotubes

Desmin is the guardian of striated muscle integrity, permitting the maintenance of muscle shape and the efficiency of contractile activity. It is also a key mediator of cell homeostasis and survival. To ensure the fine regulation of skeletal muscle processes, desmin is regulated by post-translational modifications (PTMs). It is more precisely phosphorylated by several kinases connecting desmin to intracellular processes. Desmin is also modified by O-GlcNAcylation, an atypical glycosylation. However, the functional consequence of O-GlcNAcylation on desmin is still unknown, nor its impact on desmin phosphorylation. In a model of C2C12 myotubes, we modulated the global O-GlcNAcylation level, and we determined whether the expression, the PTMs and the partition of desmin toward insoluble material or cytoskeleton were impacted or not. We have demonstrated in the herein paper that O-GlcNAcylation variations led to changes in desmin behaviour. In particular, our data clearly showed that O-GlcNAcylation increase led to a decrease of phosphorylation level on desmin that seems to involve CamKII correlated to a decrease of its partition toward cytoskeleton. Our data showed that phosphorylation/O-GlcNAcylation interplay is highly complex on desmin, supporting that a PTMs signature could occur on desmin to finely regulate its partition (i.e. distribution) with a spatio-temporal regulation.

Histone methyltransferase Dot1L recruits O-GlcNAc transferase to target chromatin sites to regulate histone O-GlcNAcylation

O-GlcNAc transferase (OGT) is the distinctive enzyme responsible for catalyzing O-GlcNAc addition to the serine or threonine residues of thousands of cytoplasmic and nuclear proteins involved in such basic cellular processes as DNA damage repair, RNA splicing, and transcription preinitiation and initiation complex assembly. However, the molecular mechanism by which OGT regulates gene transcription remains elusive. Using proximity labeling-based mass spectrometry, here, we searched for functional partners of OGT and identified interacting protein Dot1L, a conserved and unique histone methyltransferase known to mediate histone H3 Lys79 methylation, which is required for gene transcription, DNA damage repair, cell proliferation, and embryo development. Although this specific interaction with OGT does not regulate the enzymatic activity of Dot1L, we show that it does facilitate OGT-dependent histone O-GlcNAcylation. Moreover, we demonstrate that OGT associates with Dot1L at transcription start sites and that depleting Dot1L decreases OGT associated with chromatin globally. Notably, we also show that downregulation of Dot1L reduces the levels of histone H2B S112 O-GlcNAcylation and histone H2B K120 ubiquitination in vivo, which are associated with gene transcription regulation. Taken together, these results reveal that O-GlcNAcylation of chromatin is dependent on Dot1L.

Beneficial Effects of O-GlcNAc Stimulation in a Young Rat Model of Sepsis: Beyond Modulation of Gene Expression.

The young population, which is particularly at risk of sepsis, is, paradoxically, rarely studied. Acute stimulation of O-GlcNAcylation, a post-translational modification involved in metabolic regulation, cell survival and stress response, is beneficial in young rats with sepsis. Considering that sepsis impacts the gene expression profile and that O-GlcNAcylation is a regulator of transcription, the aims of this study are to (i) unveil beneficial mechanisms of O-GlcNAcylation and (ii) decipher the relationship between O-GlcNAcylation and transcription during sepsis. Endotoxemic challenge was induced in 28-day-old male rats using a lipopolysaccharide injection (E. coli O111:B4, 20 mg·kg−1) and compared to control rats (NaCl 0.9%). One hour after, rats were assigned to no therapy or fluidotherapy (NaCl 0.9%, 10 mL.kg−1) ± NButGT (10 mg·kg−1) to stimulate O-GlcNAc levels. Cardiac O-GlcNAcylation levels were evaluated via Western blot and gene transcription using 3′ SRP analysis. Lipopolysaccharide injection favorizes inflammatory state with the overexpression of genes involved in the NF-κB, JAK/STAT and MAPK pathways. NButGT treatment increased cardiac O-GlcNAcylation levels (p < 0.05). Yet, the mRNA expression was not impacted two hours after fluidotherapy or NButGT treatment. In conclusion, O-GlcNAc stimulation-induced beneficial effects are not dependent on the gene expression profile at the early phase of sepsis.

Involvement of O-GlcNAcylation in vascular damage during septic shock

Septic shock is characterized by a systemic inflammatory response syndrome (SIRS) causing hypotension and systemic hypoperfusion leading to altered cellular metabolism. This pathology is accompanied by vascular leakage due to endothelial damage. O-GlcNAcylation is a post-translational modification involved in the stress response. Recently, stimulation of O-GlcNAcylation has been shown to improve cardiovascular function in several murine models of septic shock. An O-GlcNAcylome study identified VE-cadherin and ZO-1 as potential interesting target, which are two major proteins involved in vascular leakage. We hypothesize that stimulation of O-GlcNAcylation may be involved in the regulation of vascular damage during early septic shock. Septic shock was induced in rats ( n = 10–12) by cecal ligation and punction (CLP). At 8 h and 22 h post-surgery, CLP rats receive fluid therapy with or without OGA inhibitor, NButGT (10 mg/kg) to increase O-GlcNAcylation levels. Cardiovascular functions were assessed and aortic arteries and heart were harvested and analyzed at 24 h post-surgery. Expression levels of O-GlcNAcylation, VE-cadherin and ZO-1 were assessed by western blot. The localization of VE-cadherin and ZO-1 was analyzed in the aorta. Evaluation of O-GlcNAcylation on VE-cadherin and ZO-1 were performed by immunoprecipitation and wheat germ agglutinin (WGA) gel. The results of the involvement of NButGT treatment on CLP rats show a cardiovascular improvement through mean arterial pressure (SHAM: 101.2 ± 1.65; CLP: 87.8 ± 4.67; NButGT: 102.9 ± 3.42; n = 10–12; P < 0.05). VE-Cadherin expression in the heart seems to be decreased in CLP group and this expression was significantly reversed with the NButGT treatment (SHAM: 1.00 ± 0.05; CLP: 0.86 ± 0.11; NButGT: 1.27 ± 0.10; n = 6–12; P < 0.05). WGA gel has made it possible to confirm that VE-Cadherin is O-GlcNAcylated. This study will allow us to better understand the mechanisms involved in vascular damage following the improvement of cardiovascular functions by O-GlcNAcylation stimulation in our septic shock model.

O-GlcNAcylation: The Underestimated Emerging Regulators of Skeletal Muscle Physiology.

O-GlcNAcylation is a highly dynamic, reversible and atypical glycosylation that regulates the activity, biological function, stability, sublocation and interaction of target proteins. O-GlcNAcylation receives and coordinates different signal inputs as an intracellular integrator similar to the nutrient sensor and stress receptor, which target multiple substrates with spatio-temporal analysis specifically to maintain cellular homeostasis and normal physiological functions. Our review gives a brief description of O-GlcNAcylation and its only two processing enzymes and HBP flux, which will help to better understand its physiological characteristics of sensing nutrition and environmental cues. This nutritional and stress-sensitive properties of O-GlcNAcylation allow it to participate in the precise regulation of skeletal muscle metabolism. This review discusses the mechanism of O-GlcNAcylation to alleviate metabolic disorders and the controversy about the insulin resistance of skeletal muscle. The level of global O-GlcNAcylation is precisely controlled and maintained in the “optimal zone”, and its abnormal changes is a potential factor in the pathogenesis of cancer, neurodegeneration, diabetes and diabetic complications. Although the essential role of O-GlcNAcylation in skeletal muscle physiology has been widely studied and recognized, it still is underestimated and overlooked. This review highlights the latest progress and potential mechanisms of O-GlcNAcylation in the regulation of skeletal muscle contraction and structural properties.

Structure-based discovery and development of novel O-GlcNAcase inhibitors for the treatment of Alzheimer's disease

The neurofibrillary tangles (NFTs) formed from hyperphosphorylation of tau protein are closely associated with Alzheimer's disease (AD). O-GlcNAcylation of tau can negatively regulate hyperphosphorylation and the O-GlcNAcase (OGA) catalyzes the removal of O-linked β-N-acetylglucosamine (O-GlcNAc) from tau protein. Therefore, preventing tau hyperphosphorylation by increasing the levels of tau O-GlcNAcylation via OGA inhibitors could be a promising approach. Based on Thiamet-G, a potent OGA inhibitor, and its binding mode to OGA, a novel OGA inhibitor scaffold bearing three parts was designed and hit compound 7j was successfully identified via extensive exploring. Further chemical optimization and diversification of the 7j structure resulted in compound 39 which possesses excellent OGA inhibition, no cytotoxicity, and has good pharmacokinetic properties. In acute AD model mice, 39 was more effective than Thiamet-G in inhibiting OGA activity attributable to its better blood-brain barrier permeability. In addition, 39 restored the cognitive function in mice and reduced amyloid-β (Aβ) concentrations to a greater extent than Thiamet-G. Molecular docking studies demonstrated that 39 was well associated with OGA through H-bonds and hydrophobic interaction. Together, these findings suggest that 39 was promising as a potent OGA inhibitor in the treatment of AD.

The interaction of O-GlcNAc-modified NLRX1 and IKK-α modulates IL-1β expression in M1 macrophages.

NOD-like receptor (NLR)X1 (NLRX1) is a negative regulator of inflammation by inhibiting nuclear factor-κB (NF-κB) signaling and downstream pro-inflammatory factors. However, its post-translational modification and how it participates in regulating the inflammatory responses in macrophages are still unclear. Here, we found that NLRX1 was modified with O-linked N-acetylglucosamine (O-GlcNAc). The interaction and co-localization between NLRX1 and O-GlcNAc transferase (OGT) was validated by co-immunoprecipitation and confocal microscopy analysis, and the nucleotide-binding domain (NBD) region of NLRX1 was required for its interaction with OGT. NLRX1 protein increased significantly after treatment with a high dose of OGT inhibitor OSMI-1. Elevated O-GlcNAcylation level promoted NLRX1 ubiquitination and decreased NLRX1 stability proved by ubiquitination and cycloheximide (CHX) chase experiments, and enhanced the interaction between NLRX1 and inhibitor of nuclear factor kappaB kinase-α (IKK-α), thus reducing the expression of inflammatory cytokine IL-1β in M1 macrophages. Together, our results indicate that the interaction between NLRX1 and O-GlcNAcylation coordinates and modulates the inflammatory process in macrophages.

Treadmill running increases the calcium sensitivity of myofilaments in diabetic rats.

The cardiovascular benefits of regular exercise are unequivocal, yet patients with type 2 diabetes respond poorly to exercise due to a reduced cardiac reserve. The contractile response of diabetic cardiomyocytes to β-adrenergic stimulation is attenuated, which may result in altered myofilament calcium sensitivity and posttranslational modifications of cardiac troponin I (cTnI). Treadmill running increases myofilament calcium sensitivity in nondiabetic rats, and thus we hypothesized that endurance training would increase calcium sensitivity of diabetic cardiomyocytes and alter site-specific phosphorylation of cTnI. Calcium sensitivity, or pCa50, was measured in Zucker diabetic fatty (ZDF), nondiabetic (nDM), and diabetic (DM) rat hearts after 8 wk of either a sedentary (SED) or progressive treadmill running (TR) intervention. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Specific phospho-sites on cTnI and O-GlcNAcylation were quantified by immunoblot and total protein phosphorylation by fluorescent gel staining (ProQ Diamond). The novel finding in this study was that training increased pCa50 in both DM and nDM cardiomyocytes (P = 0.009). Phosphorylation of cTnI amino acid residues Ser23/24, a crucial protein kinase A site, and Threonine (Thr)144 was lower in DM hearts, but there was no effect of training on site-specific phosphorylation. In addition, total phosphorylation and O-GlcNAcylation levels were not different between SED and TR groups. These findings suggest that regular exercise may benefit the diabetic heart by specifically targeting myofilament contractile function.NEW & NOTEWORTHY We examined the effects of training on the myofilament calcium in diabetic rat hearts. After 8 wk of treadmill running, both nondiabetic and diabetic cardiomyocytes had increased myofilament calcium sensitivity compared with their sedentary counterparts, but there was no effect of training on the phosphorylation or O-GlcNAcylation status of myofilament proteins measured in this study. These data highlight one potential mechanism capable of reversing, in part, reduced cardiac reserve in the diabetic heart.

O-GlcNAcylation and Regulation of Galectin-3 in Extraembryonic Endoderm Differentiation.

The regulation of proteins through the addition and removal of O-linked β-N-acetylglucosamine (O-GlcNAc) plays a role in many signaling events, specifically in stem cell pluripotency and the regulation of differentiation. However, these post-translational modifications have not been explored in extraembryonic endoderm (XEN) differentiation. Of the plethora of proteins regulated through O-GlcNAc, we explored galectin-3 as a candidate protein known to have various intracellular and extracellular functions. Based on other studies, we predicted a reduction in global O-GlcNAcylation levels and a distinct galectin expression profile in XEN cells relative to embryonic stem (ES) cells. By conducting dot blot analysis, XEN cells had decreased levels of global O-GlcNAc than ES cells, which reflected a disbalance in the expression of genes encoding O-GlcNAc cycle enzymes. Immunoassays (Western blot and ELISA) revealed that although XEN cells (low O-GlcNAc) had lower concentrations of both intracellular and extracellular galectin-3 than ES cells (high O-GlcNAc), the relative secretion of galectin-3 was significantly increased by XEN cells. Inducing ES cells toward XEN in the presence of an O-GlcNAcase inhibitor was not sufficient to inhibit XEN differentiation. However, global O-GlcNAcylation was found to decrease in differentiated cells and the extracellular localization of galectin-3 accompanies these changes. Inhibiting global O-GlcNAcylation status does not, however, impact pluripotency and the ability of ES cells to differentiate to the XEN lineage.

Increased O-GlcNAcylation promotes IGF-1 receptor/PhosphatidyI Inositol-3 kinase/Akt pathway in cervical cancer cells

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification on serine and threonine residues of cytosolic, nuclear and mitochondrial proteins. O-GlcNAcylation level is regulated by OGT (O-GlcNAc transferase), which adds GlcNAc on proteins, and OGA (O-GlcNAcase), which removes it. Abnormal level of protein O-GlcNAcylation has been observed in numerous cancer cell types, including cervical cancer cells. In the present study, we have evaluated the effect of increasing protein O-GlcNAcylation on cervical cancer-derived CaSki cells. We observed that pharmacological enhancement of protein O-GlcNAcylation by Thiamet G (an inhibitor of OGA) and glucosamine (which provides UDP-GlcNAc substrate to OGT) increases CaSki cells proliferation, migration and survival. Moreover, we showed that increased O-GlcNAcylation promotes IGF-1 receptor (IGF1R) autophosphorylation, possibly through inhibition of protein tyrosine-phosphatase 1B activity. This was associated with increased IGF-1-induced phosphatidyl-Inositol 3-phosphate production at the plasma membrane and increased Akt activation in CaSki cells. Finally, we showed that protein O-GlcNAcylation and Akt phosphorylation levels were higher in human cervical cancer samples compared to healthy cervix tissues, and a highly positive correlation was observed between O-GlcNAcylation level and Akt phosphorylation in theses tissues. Together, our results indicate that increased O-GlcNAcylation, by activating IGF1R/ Phosphatidyl inositol 3-Kinase (PI-3K)/Akt signaling, may participate in cervical cancer cell growth and proliferation.

O-GlcNAcylation of myocyte-specific enhancer factor 2D negatively regulates insulin secretion from pancreatic β-cells

Patients with type 2 diabetes often exhibit impairments in both glucose-induced insulin secretion (GIIS) and incretin-induced insulin secretion (IIIS). These phenotypes are associated with altered glucose metabolism in pancreatic β-cells, although the molecular mechanisms remain unclear. Here, we used MIN6-K8 pancreatic β-cell lines as a model to examine the effect of O-linked N-acetylglucosamine glycosylation (O-GlcNAcylation), a glucose-induced protein posttranslational modification, on insulin secretion. O-GlcNAcylation was enhanced in high-glucose-treated MIN6-K8 cells, and high levels of O-GlcNAcylation attenuated PKA-dependent phosphorylation, suggesting that the two protein modifications may compete with each other. Immunoprecipitation proteomic analysis identified six candidate proteins that were O-GlcNAcylated by high-glucose treatment, whereas the O-GlcNAcylations were removed by treatment with an incretin mimetic, exendin-4. Among these proteins, knockdown of myocyte enhancer factor 2D (Mef2d) enhanced insulin secretion, and high-glucose treatment increased the level of O-GlcNAcylation of Mef2d in MIN6-K8 cells. Furthermore, knockout of Mef2d promoted GIIS in MIN6-K8 cells, whereas adenovirus-mediated rescue of Mef2d decreased GIIS in the knockout cells. These results suggest that Mef2d negatively regulates insulin secretion through O-GlcNAcylation.